Biological molecules are often coupled to other molecules or compounds for use in bioanalytical or biopharmaceutical applications. The covalent combination of a biological molecule and another molecule or compound is generally referred to as a "conjugate." For example, the term "immunoconjugate" generally refers to a conjugate composed of an antibody or antibody fragment and some other molecule such as a label compound (e.g., a fluorophore), a binding ligand (e.g., a biotin derivative), or a therapeutic agent (e.g., a therapeutic protein or toxin). These particular conjugates are useful in reporting the presence of the antibody, binding or capturing the antibody, and targeting the delivery of a therapeutic agent to a specific site, respectively.
Conjugates are prepared by covalently coupling one of the conjugate components to the other. For instance, an immunoconjugate may be prepared by coupling a label compound, a binding ligand, or a therapeutic agent to an antibody or antibody fragment. Often the coupling involves the use of a linker compound or molecule which serves to join the conjugate components. For example, a typical immunoconjugate is composed of a biotin component covalently coupled to an antibody component through a linker. Because the linker is typically chosen to provide a stable coupling between the two components, the usefulness of the conjugate is generally limited by the stability of the linkage between the conjugate components--that is, the greater the stability of the linkage between the components of a conjugate, the more useful and effective the conjugate. Depending upon a conjugate's use, a wide variety of conjugates may be prepared by coupling one conjugate component to another via a linker. Virtually an endless number of combinations of a biological molecule coupled to a label compound, binding ligand or therapeutic agent have been joined to create conjugates suitable for a particular purpose or need.
An example of a useful and widely employed class of conjugates include biotin conjugates. Biotin is a naturally occurring vitamin which has an extremely high binding affinity (K.sub.d .apprxeq.10.sup.-15 M.sup.-1) for avidin and streptavidin. Because of the affinity of biotin for avidin, biotin-containing conjugates have been widely used in bioanalytical procedures including immunoassays, affinity chromatography, immunocytochemistry, and nucleic acid hybridization (see, e.g., Green, Adv. Protein Chem. 29:85, 1975; Wilchek and Bayer, Anal. Biochem. 171:1, 1988; Wilchek and Bayer, Meth. Enzymol. 184:5, 1990). Bioanalytical assays often take advantage of the high binding affinity of biotin for avidin through the covalent coupling of biotin to one of the assay components. To this end, biotin may be covalently coupled to many different types of molecules, including proteins, such as antibodies, antibody fragments, enzymes and hormones; nucleic acids such as oligonucleotides and a nucleic acid probes; and smaller molecules such as drugs or other similar compounds. Moreover, in some applications biotin may be coupled to a solid phase or support.
The covalent coupling of biotin to another molecule involves bond formation through chemical reaction between suitable chemical functional groups. For the coupling of biotin to a molecule such as an antibody or enzyme, a reactive biotin derivative is generally used. Reactive biotin derivatives for conjugation may readily be prepared from biotin, and are most commonly carboxylic acid derivatives or, in some cases, nucleophilic derivatives such as amine or hydrazide derivatives. Common reactive biotin derivatives include reactive biotin esters such as an N-hydroxysuccinimide (NHS) ester. For example, biotin NHS esters may be conveniently attached to proteins and peptides through a free amino group, such as the epsilon amino group on lysine residues. Other reactive biotin derivatives include nucleophilic derivatives, such as biotin hydrazide, which may be conjugated to glycoproteins through aldehyde groups generated by oxidation of their carbohydrate groups. Reactive biotin derivatives are commercially available from a variety of sources including Sigma (St. Louis, Mo.), Pierce (Rockford, Ill.), and Molecular Probes (Eugene, Oreg.). Many of these biotin derivatives contain various chemical groups between the biotin moiety and the reactive group. Methods of conjugating biotin derivatives to proteins have been described in numerous publications (see, e.g., Harlow and Lane, Antibodies. A Laboratory Manual, NY: Cold Spring Harbor Laboratory, 1988, pp. 340-341; O'Shannessy and Quarles, J. Imnmunol. Methods 99:153, 1987; O'Shannessy et al., Immunol. Letters 8:273, 1984; Rose et al., Bioconjug Chem. 2: 154, 1991).
In addition to biotin, other compounds are commonly coupled to biological molecules for use in bioanalytical procedures. Typically, these compounds are useful in labeling the biological molecule for detection purposes. Common labeling compounds include fluorescent dyes, as well as ligands for binding to their respective binding partners. Examples of common fluorescent dyes used for this purpose include fluorescein and rhodamine, and examples of ligands for binding to their binding partners include drug compounds such as digoxigenin and .beta.-lactam antibiotics. Numerous other compounds suitable for use as labels in specific binding techniques have also been described in the literature. Like biotin, these compounds are generally derivatized to contain functional groups that react readily with the biological molecule. For example, fluorescein isothiocyanate is a reactive fluorescein derivative which may readily be conjugated to proteins through their sulfhydryl groups.
Effective conjugation of a compound, such as biotin or a fluorescent dye, to a biological molecule generally requires that the resulting labeled conjugate retain the bioactivity of the biological molecule. A conjugate may have only limited utility if, upon coupling, the functional activity of the biological molecule is diminished or lost. For example, for an antibody conjugate, retention of antigen binding activity (immunoreactivity) is of foremost importance. Because some antibodies lose immunoreactivity upon labeling of their free amino groups, presumably due to the presence of these groups in the antigen binding site of the antibody (see Harlow and Lane), the site or sites at which a label is attached to a biological molecule is of considerable importance. Similarly, some enzymes contain free amino groups in their active sites which, upon their use as a labeling site, may result in a loss of enzymatic activity. Many enzymes also contain sulfhydryl groups in their active sites and are inactivated by labeling with sulfhydryl-reactive compounds such as fluorescein isothiocyanate.
In addition to retaining bioactivity, the stability of the conjugate with respect to linkage of the compound to the biological molecule is very important. For example, loss of a label from a conjugate typically results in the loss of ability to follow the conjugate in a bioanalytical procedure. In an attempt to provide stable linkages, conjugates are often coupled through amide and hydrazone bonds. Amide linkages are formed by reaction between an amino group and a carboxylic acid group, and hydrazone linkages result from reaction of a carbonyl group (such as an aldehyde group) and a hydrazine group. The relatively high stability of these linkages has led to their wide use in conjugation techniques (see, e.g., O'Shannessy et al., 1984; Reeves, in The Chemistry of the Carbonyl Group, S. Patai (ed.), NY: Interscience, 1966, pp. 567-619). However, while such conjugates may be stable at neutral pH, these conjugates become unstable at acid pH (Hurwitz et al., J. Applied Biochem. 2:23, 1980; Kaneko et al., Bioconj. Chem. 2:133, 1991). In fact, investigators have even exploited the pH-dependent stability of the hydrazone bond to design antibody-drug conjugates that retain the drug in the generally neutral pH environment of the peripheral circulation, and release the drug when the conjugate is exposed to an acidic environment such as is found in certain cell organelles.
Because of the perceived stability of hydrazone and amide bonds, the usual solution to the problem of activity loss by a hydrazone- or amide-linked labeled conjugate is to use more of the conjugate (i.e., to re-titer the conjugate) or to prepare a "fresh" conjugate. For example, where loss of functional activity of a biotin-antibody conjugate has been observed, it has been generally assumed that this loss is due to a loss in immunoreactivity of the antibody portion of the conjugate. This assumption, however, may be invalid in many instances. Rather, depending upon storage conditions, the immunoreactivity of the antibody portion of the conjugate may be undiminished, and the effectiveness of the conjugate is compromised by use of a linker that is unstable at the storage and/or use conditions.
Accordingly, there is a need in the art for improved linkages for conjugating a biological molecule with, for example, a label compound, binding ligand or agent, or therapeutic agent. Such linkages should have enhanced stability such that the effectiveness of the conjugate is not diminished through storage and/or use conditions. The present invention fulfills these needs and provides further related advantages.